Cystic fibrosis (CF) is caused by mutation in the single gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane Cl- channel. Despite extensive study, there are significant gaps in our understanding of how CFTR is synthesized and processed and how CFTR is regulated and functions at the apical membrane. CFTR associates with a number of proteins that facilitate its trafficking or function, but our understanding of these interactions and how they are altered in CF is relatively poor. We find that the actin binding proteins filamin A (FLN-A) and FLN-B associate directly with residues 1-25 of human CFTR. This interaction enhances receptor-stimulated activation of CFTR and a known disease-causing mutation in the N-terminus of CFTR (S13F) decreases the affinity of the interaction. In addition, the half-life of S13F CFTR is significantly decreased when compared to CFTR proteins that can bind FLN. We also find that a novel sorting nexin, SNX27, associates with CFTR. SNX27 accumulates on subapical endosomes and RNA interference-mediated depletion of SNX27 significantly decreased the levels of cell surface-associated CFTR. Since FLNs and SNX27 associate with CFTR at the cell surface or in endosomal compartments, we hypothesize that these novel CFTR-interacting proteins modulate aspects of CFTR internalization and recycling in polarized cells. Therefore, we propose to fully characterize the biological significance of the CFTR-FLN and CFTR-SNX27 interactions using biochemical, cellular, and functional assays in airway epithelial cells. Our data suggest that CFTR is tethered to the actin cytoskeleton via two distinct linkages - an N-terminal interaction with FLN and C-terminal interaction with actin-associated PDZ proteins. Therefore we will test the hypothesis that these two cytoskeletal anchors work in concert to stabilize CFTR at the apical membrane of polarized cells. The characterization of protein interactions that modulate CFTR trafficking, stability and/or function provide one prospect for new therapies for CF and will increase our understanding of the trafficking and regulation of this complex epithelial ion channel. Lay Summary: Mutations in CFTR cause the lethal childhood disease, cystic fibrosis;our experiments address the question of how other proteins that bind CFTR alter its function in the human lung.